Asthma is a chronic respiratory disorder that afflicts hundreds of millions of people throughout the world [Drazen and Weiss, Nature 418:383-384 (2002)]. Though the occurrence of this respiratory disorder has been noted for over two thousand years, during the past twenty years industrialized nations have experienced an increase in asthma sufferers that approaches epidemic proportions [Umetsu et al., Nature Immunology 3:715-720 (2002)]. Indeed, 10-20% of the population of industrialized countries currently suffers from asthma. Not surprisingly, the dramatic increase in the number of asthmatics in industrialized nations has resulted in a concomitant expenditure of resources to treat this condition [Umetsu et al., Nature Immunology 3:715-720 (2002)]. Despite this strong commitment, to date the treatments employed only control the symptoms.
Asthma is characterized by life-threatening attacks due to episodic obstructions to, or abnormal narrowing of the airways in response to otherwise innocuous stimuli [Drazen and Weiss, Nature 418:383-384 (2002)]. Common symptoms of asthma include recurrent episodes of coughing, wheezing and breathlessness. The immediate cause for the thickening of the airway walls, smooth muscle contraction, and narrowing of the airways observed in asthmatics is an inflammation mediated by T-cells [Van Eerdewegh et al., Nature 418:426-430 (2002)]. Both genetic and environmental factors play key roles in inducing this T-cell-mediated inflammation, though the actual mechanism has yet to be delineated. What is known is that asthmatics have a genetic predisposition for the disease, and environmental factors serve to either trigger or protect against this immunological dysregulation [Umetsu et al., Nature Immunology 3:715-720 (2002)].
Recently, the gene encoding a membrane anchored protein known as ADAM33 has been shown to be linked to asthma by positional cloning in an outbred population [Van Eerdewegh et al., Nature 418:426-430 (2002)]. ADAM33 is a member of the “A Disintegrin And Metalloprotease” (ADAM) family of proteins which comprises over thirty such proteins, including the well characterized ADAM17, the TNF-α converting enzyme (TACE) [Cross et al., J. Am. Chem. Soc. 124:11004-11007 (2002); Schlondorff and Blobel, J. Cell Sci., 112:3603-3617 (1999); Black, Intern.J. Biochem. Cell Biol 34:1-5 (2002); U.S. Pat. No. 5,830,742]. The ADAM family of proteins is a class of type-I transmembrane proteins that share a unique domain structure composed of a signal sequence, a pro domain, a metalloprotease/catatlytic domain, a disintegrin domain, a cysteine-rich domain, an epidermal growth factor-like domain, a transmembrane and a cytoplasmic domain.
U.S. Pat. No. 6,420,154 B1 discloses a human nucleic acid sequence that subsequently was shown to encode ADAM33, along with the corresponding amino acid sequence. Others also have disclosed human and mouse ADAM33 nucleic acid and amino acid sequences [Yoshinaka et al., Gene 282:227-236 (2002); Gunn et al., BMC Genetics 3:2 1-8, (2002)]. However, little specific information has been provided regarding the catalytic activity of ADAM33. Moreover, heretofore, the ADAM33 protein domains, including the catalytic domain, had not been specifically delineated and isolated.
Due to its genetic linkage to asthma, ADAM33 has become a promising target protein for use in identifying pharmaceuticals to treat asthma [Shapiro and Owen, N Engl J Med 347:936-938 (2002)]. Structure based drug design is one way to optimize the success of such drug discovery. However, use of this powerful methodology requires the three-dimensional structure of the target protein and, heretofore, little to no information has been provided regarding the three-dimensional structure of ADAM33. This is in sharp contrast with other Zinc dependent metalloproteases such as Adamalysin II, [Gomis-Ruth et al., Protein Science 7:283-292 (1998)] and TACE, [Letavic et al., Biorgan. & Medic. Chem Lett. 12:1387-1390 (2002); WO9940182] for which three-dimensional structures have been determined. Indeed, the current inability to generate X-ray diffractable crystals of ADAM33 and/or of its catalytic domain has greatly hampered efforts for obtaining the requisite structural information necessary to perform structure based drug design on this protease.
Therefore, there is a need to define the nucleic acid and amino acid sequences of the catalytic domain of ADAM33. Moreover, there is a need to prepare nucleic acid constructs that encode the ADAM33 catalytic domain. In addition, there is a need to design purification procedures that lead to the preparation of isolated active ADAM33 protein and/or fragments thereof. Furthermore, there is a need to obtain ADAM33 protein samples that are amenable to forming homogenous crystals for X-ray crystallization analyses. In addition, there is a need to obtain X-ray diffractable crystals of the ADAM33 catalytic domain of sufficient quality for X-ray crystallization analyses. Moreover, there is a need to obtain crystals of the ADAM33 catalytic domain that are amenable to ligand exchange. Furthermore, there is a need to provide methods for identifying inhibitors of ADAM33 through structure based drug design.
The citation of any reference herein should not be construed as an admission that such reference is available as “prior art” to the instant application.